Abstract The goals of this project are to develop 19F-nanoprobes targeted to biomarkers of macrophage polarization, and to enable quantification of multiple polarization markers in in vivo as a measure of macrophage functional state. The plasticity of macrophages can place them along a spectrum of functional states, with the M1 phenotype at one pole driving inflammation and the M2 phenotype at the other pole suppressing immune activity. With increased interest in disease therapies targeting the immune system, it is vitally important to be able to assess if the immune population of macrophages is aiding or hindering the disease, and identifying functional states is key to that understanding. Recent discoveries have revealed that macrophages can play dichotomous roles in disease; they can promote resolution or facilitate progression dependent on their functional state. For example, in cancer, tumor associated macrophages (TAMs) that are M2-like can support tumor progression, while M1 function can be tumor killing. While, in atherosclerosis, excessive M1 activity can induce dangerous plaque rupture, while M2 function can promote plaque stability. Macrophage subtypes are typically identified by immunohistochemistry of extracted tissues. But biopsy is not always possible, and moreover, suffers from inability to accurately report on heterogeneous distributions of cells. There is a great, unmet need for a noninvasive method to comprehensively assess macrophage functional state in vivo. While positron emission tomography (PET) is frequently used to visualize molecular biomarkers, PET can only assess a single marker at a time. The complexity of macrophage polarization is such that a single marker cannot define their function. The ability to quantify multiple markers along the polarization spectrum would be a major advance for measuring macrophage phenotype in vivo. We propose to develop 19F-MRI for multiplexed imaging of macrophage polarization markers and will synthesize targeted nanoprobes for this purpose. We will prepare a panel of 19F-nanoprobes targeted to M1 and M2 markers (CD206, CD86, CD40, SR-A) using copper-free click chemistry to attach targeting molecules to perfluorocarbon nanoparticles. We will validate the targeting of these nanoprobes in cultured cells, and examine their effect on macrophage biology using flow cytometry, cytokine, toxicity, and gene expression assays. We will also validate targeting in a mouse model of acute inflammation (lipopolysaccharide-infused Matrigel placed subcutaneously). We will then synthesize these nanoprobes with different perfluorocarbon cores, to provide each with a unique MRI signature that can be discriminated in 19F-MRI. The animal studies will be repeated for mixtures of nanoprobes targeted to different polarization markers and we use multiplexed imaging to quantify the amount of each type of nanoprobe in vivo as a measure of the corresponding polarization marker. The success of this work establishes a new paradigm for the use of targeted 19F-MRI to assess macrophage functional states in vivo.